Immunoaffinity gel detection column for detecting gentamicin and preparation method thereof

A technology of gel detection and gentamicin, which is applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of the influence and limitation of test strips, and achieve the effect of convenient operation, high selectivity, and elimination of the influence of sample matrix.

Inactive Publication Date: 2016-03-23
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection of test strips is also susceptible to the influence of the sample matrix, which is limited in the detection of different food samples

Method used

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  • Immunoaffinity gel detection column for detecting gentamicin and preparation method thereof
  • Immunoaffinity gel detection column for detecting gentamicin and preparation method thereof
  • Immunoaffinity gel detection column for detecting gentamicin and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Purification of gentamicin antiserum

[0031] Using ProteinA-Sepharose4B as affinity chromatography medium to purify gentamicin antiserum can obtain near-purity specific antibody at one time. The specific operation steps are (1) Equilibration: flush the pipeline with equilibration buffer (0.2mol / L phosphate buffer) and equilibrate the column until the baseline is stable. (2) Sample loading: dilute the gentamycin-specific antiserum to an equal volume with an equilibration buffer and apply to the column. (3) Impurity washing: wash with equilibration buffer until the ultraviolet absorption peak of the impurity protein appears, then continue to wash until the baseline is stable. (4) Elution collection: the specific IgG antibody was eluted with 0.1 mol / L glycine buffer solution at pH 2.7. When the UV absorption curve shows an upward trend, collect the specific antibody. And quickly use Tris-HCl to neutralize the antibody to neutrality. (5) Capping the column: After th...

Embodiment 2

[0044] How to use gentamicin gel detection column

[0045] 1. Detection steps

[0046] (1) Adding samples: Pre-mix the diluted enzyme-labeled antigen and gentamicin sample solution with 1mL LPBS, pass through the column through the injection port, and control the flow rate to complete the flow in 1min.

[0047] (2) Wash the column: add 3mL of PBST from the injection port to wash the column, and then wash with 2mL of PBS to remove the antigen not bound to the antibody and the residual Tween20.

[0048] (3) Color development: Inhale 300 μL of the substrate solution from the bottom, react for 30 seconds, inject air with the syringe plunger, completely discharge the substrate solution, continue to develop color for 4 minutes, and observe the results.

[0049] 2. Result judgment

[0050] Visually inspect the color of the quality control layer and detection layer of the gel column. The quality control layer is obviously blue, indicating that the detection column can be used norma...

Embodiment 3

[0052] Examples of application effects of the present invention

[0053] 1. Sample processing method

[0054] Take 2mL (milk) or 2g (tissue) sample and add 2mL PBS buffer, shake at 2000r / min for 4min, then centrifuge at 5000r / min for 15min, take 1mL of supernatant, and make 10-fold dilution. Take 1mL of the final sample treatment solution, add enzyme-labeled antigen to it, pass the column for color development, and observe the color development results.

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PUM

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Abstract

The invention provides a preparation method of an immunoaffinity gel detection column capable of visually and rapidly detecting gentamicin residues in food. The method comprises the steps that agarose gel is used as a solid-phase carrier, a gentamicin antibody and the agarose gel which is activated through cyanogen bromide are coupled to prepare antibody gel used as a detection layer, an HRP antibody and the agarose gel which is activated through the cyanogen bromide are coupled to prepare HRP antibody gel used as a quality control layer, the product is placed into 1mL of solid phase extraction column, and the immunoaffinity gel detection column is prepared. The novel immunoaffinity gel detection product which detects the gentamicin residues in the food rapidly, qualitatively and semiquantitatively is developed, and the detection limit is 40 micrograms / L. The immunoaffinity gel detection column has the outstanding advantages that specificity is high, and sensitivity is good; sample pretreatment is simple; detection time is short and accuracy is high; operation is easy and convenient, and assisting of large instruments is not needed.

Description

technical field [0001] The invention belongs to the technical field of immunochemistry and residue analysis of small molecular compounds, and relates to immunochemistry, enzymology and analytical chemistry techniques, and in particular to the preparation of a gentamicin immunoaffinity gel visualization detection column. Background technique [0002] Gentamicin is an aminoglycoside antibiotic produced by the fermentation of Micromonospora crimsonosa and Micromonas aculeatus. It is easily soluble in water, alkaline and stable in nature. It mainly acts on Gram-negative bacteria, blocking bacterial protein synthesis in the whole process, including inhibiting the formation of 70S complexes in the initiation phase, interfering with DNA replication, affecting peptide chain extension, etc., changing the permeability of cell membranes, thereby playing an antibacterial and bactericidal role . Gentamicin has wide antibacterial spectrum, good curative effect and low price, so it is wid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/559G01N33/535
CPCG01N33/559G01N33/535
Inventor 生威王硕张燕王俊平胡高爽
Owner TIANJIN UNIV OF SCI & TECH
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